The underpinning of this proposal is that there is an urgent need for new pharmacotherapy of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate the brush border Na/H exchanger NHE3 under both the basal state and in the inhibited state as occurs in most diarrhea. A starting point in achieving this stimulation is to understand how NHE3, which accounts for the majority of intestinal Na absorption at least in the fasting state, is regulated both normally as part of digestive physiology and as inhibited in diarrhea. In this proposal we will expand understanding of acute NHE3 regulation by examining the mechanisms of action of several proteins that we have found both bind to the NHE3 C-terminus and regulate NHE3 activity. These proteins are 1) CaM kinase II, 2) CK2, 3) PLC3. The major hypothesis to be tested in this proposal is that NHE3 activity is determined under both basal and elevated Ca2+ conditions by coordinated and dynamic interactions with these C-terminal binding partners, which not only regulate NHE3 but also affect each others'association with and regulation of NHE3 in a coordinated manner. Aim I studies each of these proteins expressed in polarized intestinal Na absorptive cells (Caco-2) and uses transport assays correlated with biochemical approaches including siRNA and pharmacologic and molecular (dominant-negative) inhibitors with molecularly modified NHE3 or the associating proteins to determine the consequences of NHE3 physical association with these scaffolded proteins on NHE3 basal, stimulated and inhibited activity and on NHE3 complexes involved in its regulation. Aim II tests the hypothesis that a common domain in the NHE3 C-terminus with which all of the proteins in Aim I associate, represents part of "a switch" which moves NHE3 back and forth between a fully stimulated state through basal activity to inhibition, which defines NHE3 function as part of digestion. This will be examined by determining whether association with NHE3 of the proteins studied in Aim I changes in a reproducible and time dependent manner with changes in regulation of NHE3 activity;whether knockdown and pharmacologic/molecular inhibitors of each of these NHE3 associating proteins alter NHE3 association with the other interacting proteins;and the consequences of eliminating interactions with single or multiple of these associating proteins including by mutagenesis approaches. Public Health Relevance: The basis of this proposal is that there is an urgent need for new drug treatment of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate a specific intestinal sodium transport protein, the brush border Na/H exchanger NHE3, under both the basal state and in the inhibited state as occurs in most diarrheal diseases. This study examines the mechanism by which several proteins which bind to the cytoplasmic part of NHE3 affect its transport activity under basal conditions and when NHE3 is stimulated or inhibited to mimic changes which occur during digestion and in diarrheal diseases. In addition, the coordinated association with NHE3 of these proteins which all bind to a small area of NHE3, will be examined to determine if together they explain how NHE3 changes its activity during digestion and in diarrhea.